Bio-Rad PDS-1000 / He™ and Hepta™ Systems - Manual (Page 38)

Some kinds of embryogenic suspension cultures approach this ideal. In fact, it is this type of
tissue which has allowed great success in the microcarrier-mediated transformation of recalcitrant
species such as maize [see, for example, Gordon-Kamm et al., The Plant Cell, 2, 603-618 (1990)].
Most explants, however, differ from this ideal in substantive ways. It is useful, though, to keep
these characteristics in mind when choosing and preparing tissues for bombardment, whether
taken from established cultures, or recently removed from donor plants. For example, tissue dam-
age is typically most severe within the central area of bombardment where particle density is
greatest. Cultured materials can usually be arranged to minimize this effect by placing them in a
circle around the central area of bombardment. Even though some of the DNA-bearing particles
fail to enter cells, this will often increase the absolute number of transformation events recov-
ered from a single bombardment by reducing tissue damage. Similarly, explants from donor
plants should be arrayed so as to capture as large a portion of particles as possible. Newly acquired
explants can be dissected so that the cell types of interest are exposed as much as possible. The
height of the tissue above the surface of its support medium can be minimized to reduce losses
during treatment, which result from tissues with high profiles being blown about during bom-
bardment. Finally, it may be useful to reduce the size of each explant or tissue mass to more
closely approximate the geometry of suspension culture cells, in order to enhance the uniformi-
ty of selection.
Most tissues appear to vary with regard to their ability to express introduced genes fol-
lowing bombardment. For example, this effect is frequently observed as fluctuations in the fre-
quency or intensity of transient expression of ß-glucuronidase (GUS) constructions among
different tissues, or even among cells in different areas of a single explant. It can be caused
in part by unevenness in particle distribution during preparation of macrocarriers, but appar-
ently can also be caused by heterogeneity of the tissue itself. In intact explants it is not uncom-
mon to see variation in expression associated with the physiological age of the explant.
Although the causes of these phenomena are unknown, it is important to realize that such
effects exist, and can be a source of serious confounding in experiments designed to choose
tissues for use in transformation experiments, and to optimize bombardment parameters.
Optimization of Bombardment Parameters—Transient expression vs stable trans-
formation
The goal of many microcarrier bombardment experiments is to develop protocols for effi-
cient production of transgenic plants. The frequency at which stable transformation events
are recovered is the ultimate criterion by which these protocols must be measured. However,
measurement of transient expression (i.e., expression of newly introduced DNA sequences
after a relatively brief period following bombardment) provides rapid feedback, and can be an
invaluable aid in the determination of effective bombardment parameters for a specific tissue.
A precise quantitative relationship between the level or frequency of transient expression
observed in a bombarded tissue, and the frequency at which stable transformation of cells occurs
within that tissue may not exist. Transient expression is, however, a very useful indicator of the
efficiency of DNA delivery, and can help to define conditions required to deliver DNA into
specific cell layers. In this way, measurement of transient expression allows rapid determination
of a set of parameters which permit delivery of DNA into tissues of interest, and provides crit-
ical information about which parameters have the greatest effect.
Choice of reporter gene and assay procedures for optimization of bombardment must be
determined by the particular question to be addressed. Histochemical assay of ß-glucuronidase
expression in bombarded cells is currently the most commonly used measurement of the
frequency of transient expression events in bombarded tissues. It is also ideal for determin-
ing which cell types in a heterogeneous tissue are capable of expressing the introduced
sequence. This procedure is not useful, however, for determining the absolute expression
levels produced by a particular set of bombardment parameters. For such measurements, it is
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